Invasive fungal infections have increased dramatically in recent years due to increased numbers of immunocompromised individuals. In parallel, Candida glabrata has emerged as an important pathogen, largely because it is refractory to treatment with widely used azole antifungals. Echinocandins including caspofungin (CSP) and micafungin (MCF) have potent activity versus C. glabrata (MIC 0.06 ?g/ml) and were recently elevated to first-line agents for treating infections with this yeast. Rare acquired resistance (MIC 1) to these lipopeptide inhibitors of cell wall synthesis is associated with echinocandin cross-resistance and mutations in the integral membrane proteins Fks1 or Fks2. We recently described novel laboratory mutants arising at relatively high frequency that exhibit CSP reduced susceptibility (CRS; MIC = 0.12 to 0.5) but MCF increased susceptibility (MIS; MIC = 0.0005 to 0.008); clinical isolates with this phenotype were also identified. Intriguingly, FKS sequencing and gene disruption demonstrate that CRS-MIS is Fks-independent. Elucidating the CRS-MIS mechanism is important since it will (1) support and suggest new approaches to enhancing echinocandin therapy (e.g., sequential CSP-MCF, or combining MCF with an inhibitor/intermediate that mimics the CRS-MIS mutation) and (2) shed much needed light on echinocandin mechanism of action (i.e., differential susceptibilities imply that the alterd cellular component directly binds to a CSP versus MCF-specific side chain). To uncover this mechanism, two distinct genetic screens in Saccharomyces cerevisiae were employed, both implicating sphingolipid synthesis. Consequently, lipid analysis of C. glabrata CRS-MIS mutants and clinical isolates was pursued, revealing increased levels of dihydrosphingosine and phytosphingosine. Based on these data specific genes were sequenced and, indeed, mutations identified in SUR2, FEN1, SUR4, and IFA38 CRS-MIS is not limited to C. glabrata: (a) 1 of 6 Candida albicans strains tested yielded mutants (8-fold CRS, 16-fold MIS) at high frequency, and lipid analysis/DNA sequencing implicated sphingolipid pathway gene TSC13/tsc13 loss of heterozygosity; and (b) an Aspergillus nidulans basA(Sur2) mutant exhibited 2-fold CRS, 8-fold MIS. To extend these studies, we propose three Aims focusing on: (1) C. glabrata. The full complement of mutations conferring CRS-MIS will be identified, fitness assessed in the Galleria model, effects of pathways inhibitors and intermediates examined, and interaction between CRS-MIS and Fks mutations evaluated. (2) S. cerevisiae. The mechanism of sphingolipid pathway regulation by Cka2 will be explored, and the hypothesis that sphingolipid changes confer CRS-MIS through altered Fks-membrane topology will be tested. (3) C. albicans and pathogenic aspergilli. C. albicans strains exhibiting high risk CRS-MIS will be analyzed for genotypic relatedness and mechanism, and A. fumigatus and A. terreus examined for CRS-MIS capacity and mechanism.